Abstract 5059: Quantitative analysis of MT1-MMP activity on cell surface: A FRET assay model.

Abstract

Abstract Introduction: MT1-MMP plays critical roles during tumor malignancy and is one of the best-validated proteolytic enzyme targets on cancer cells. It is up-regulated in several tumor types, including breast, cervical, and ovarian cancer and is significantly associated with adverse outcome. In spite of the large proteolytic repertoire and of the robust proteolytic activity of engineered soluble forms of the enzyme, several studies indicate that soluble MT1-MMP mediated proteolysis is not sufficient per se for efficient penetration of ECM barriers. Thus, localization of the enzyme at the cell surface is essential to translate its proteolytic activity into a modification of cell function. The transmembrane nature of MT1-MMP allows it to influence extracellular remodeling of the matrix surrounding tumor cells as well as intracellular signaling events involved in cell invasion. Like virtually all cell surface proteases, quantitative assessment of MT1-MMP in its native environment has not been achieved. A mechanistic examination of MT1-MMP at the cell surface would unravel the influences of binding partners on activities, and set the stage for the development of unique, non-active site inhibitors. Methods: ΔTM MT1-MMP (a deletion mutant lacking the TM domain) was transfected transiently in COS-1 cells to generate soluble MT1-MMP. Estimation of the amount of active enzyme was done by TIMP-2 titration. WT-MT1-MMP (Wild Type) was stably transfected in MCF-7 cells which are deficient in MT1-MMP. A cell-based FRET assay was developed in a 384-well format using fluorogenic triple-helical peptide substrates (fTHPs) that are either suitable for most collagenolytic MMPs or selective for different collagenolytic MMPs to quantify protease activity. Kinetic parameters were calculated and activity comparisons were made for soluble MT1-MMP and several surface-bound MT1-MMP mutants to evaluate the importance of the cell surface and specific MT1-MMP domains on activity. Results and Conclusion: Soluble MT1-MMP was expressed, purified, and quantified by TIMP-2 titration. Kinetic parameters KM, Vmax, and kcat/KM were determined by calculation of rates of hydrolysis of fTHP-9 in a FRET assay. A high level of MT1-MMP expression in transfected cells relative to those mock transfected cells was verified by western blot. Kinetic parameters determined with WT-MT1-MMP transfected cells as compared to mock transfected cell suggested that MT1-MMP has affinity for fTHP-9. kcat/KM for the soluble enzyme was 32800 sec−1M−1, three times higher as compared to membrane tethered enzyme. Thus, the catalytic efficiency of the enzyme is different in the cell surface environment. In this study, we have developed cell based assays useful for the mechanistic and quantitative evaluation of substrates and inhibitors of MT1-MMP in its native environment as well as to rank order potential inhibitors. Citation Format: Sonia Pahwa, Gregg Fields. Quantitative analysis of MT1-MMP activity on cell surface: A FRET assay model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5059. doi:10.1158/1538-7445.AM2013-5059